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Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL-ALSOFAST Experiment with Delayed Decoupling.

ABSTRACT: Current biological research increasingly focusses on large human proteins and their complexes. Such proteins are difficult to study by NMR spectroscopy because they often can only be produced in higher eukaryotic expression systems, where deuteration is hardly feasible. Here, we present the XL-ALSOFAST-[13 C,1 H]-HMQC experiment with much improved sensitivity for fully protonated high molecular weight proteins. For the tested systems ranging from 100 to 240?kDa in size, 3-fold higher sensitivity was obtained on average for fast relaxing signals compared to current state-of-the-art experiments. In the XL-ALSOFAST approach, non-observed magnetisation is optimally exploited and transverse relaxation is minimized by the newly introduced concept of delayed decoupling. The combination of high sensitivity and superior artefact suppression makes it ideal for studying inherently unstable membrane proteins or for analysing therapeutic antibodies at natural 13 C abundance. The XL-ALSOFAST and delayed decoupling will therefore expand the range of biomolecular systems accessible to NMR spectroscopy.

SUBMITTER: Roßler P

PROVIDER: S-EPMC7589290 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL-ALSOFAST Experiment with Delayed Decoupling.

Rößler Philip P Mathieu Daniel D Gossert Alvar D AD

Angewandte Chemie (International ed. in English) 20200826 43

Current biological research increasingly focusses on large human proteins and their complexes. Such proteins are difficult to study by NMR spectroscopy because they often can only be produced in higher eukaryotic expression systems, where deuteration is hardly feasible. Here, we present the XL-ALSOFAST-[<sup>13</sup> C,<sup>1</sup> H]-HMQC experiment with much improved sensitivity for fully protonated high molecular weight proteins. For the tested systems ranging from 100 to 240 kDa in size, 3-f ...[more]

PMID: 32743971

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Project description:Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4'-fluoro-[1,1'-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices [Formula: see text]2, [Formula: see text]3 and the very beginning of [Formula: see text]5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach.

Dataset Information

Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL-ALSOFAST Experiment with Delayed Decoupling.

Publications

Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL-ALSOFAST Experiment with Delayed Decoupling.

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